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Presentation of Quiz #34

A patient is a 54 year-old male (post-transplant, liver - 14 months before) from the United States who has traveled throughout the world as a professional consultant (Europe, Asia, Central and South America, Australia, New Zealand). He was diagnosed as having diarrhea, cough, and general malaise. He had a history of vague health problems since the transplant, but did not seek medical attention for several weeks. The first stool specimen was submitted to the laboratory. After examination of the concentration sediment and permanent stained smears from all three specimens, the following objects were seen. Please comment on the identification of the structures seen.

The structure on the left measures approximately 16 μm and the structures on the right measure approximately 65 μm. The structure on the left is stained with the standard Wheatley's modification of the Gomori trichrome, while the structure on the right is a wet preparation of the sedimentation concentration.

After submission of the other two stool specimens, it was also decided to perform special stains for the coccidia and microsporidia. The following things were seen on the stained smears. Structures on the left measured ~ 4-6 μm, while those on the right measure ~1.5 μm. Please comment on the objects seen below.

Modified Acid-Fast Stain (Left) and Modified Trichrome Stain (Right)

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Answer and Discussion of Quiz #34

Answers to Questions: The images presented after the first stool specimen represent artifact material. The structure on the left mimics an Entamoeba coli cyst; however, this structure is not an organism, but an artifact. The structure on the right tends to resemble helminth eggs, but they are vegetable cells and not parasites. Unfortunately, even when structures measure within the correct size range, the morphology may not be correct or accurate enough to identify the structure as a true parasite.

The two images seen when special stains were performed provide excellent examples of Cryptosporidium spp. oocysts (on the left) and microsporidial spores (on the right), probably Enterocytozoon bieneusi or Encephalitozoon intestinalis.

Comments on the Patient: Because this patient is a post-transplant (liver) patient, and has been and continues to be immunosuppressed, continuous diarrhea with negative O&P examinations should suggest the use of special stains for the coccidia (modified acid-fast stains) or microsporidia (modified trichrome stains). These diagnostic options are important, particularly if the patient is immunosuppressed and continues to be symptomatic with diarrhea.

Comments on Diagnostic Methods: Although there are immunoassays available for the diagnosis of cryptosporidiosis (EIA, FA, cartridge formats), many laboratories continue to use the modified acid-fast stain. It is important to remember that the destaining (acid) step should be performed quickly and the acid content should not be too high. If the percentage is above 3% acid, then too much color may be removed from the oocysts. Color loss can be a definite problem for the identification of Cyclospora cayetanensis (modified acid-fast variable), but is less of a problem with Cryptosporidium.  Many laboratories are using this stain with a 1% acid destain step. Both Cryptosporidium and Cyclospora will look fine using this approach. However, regardless of the destain step, there will generally be some of the C. cayetanensis oocysts that may appear pale pink or even colorless. It is also recommended that these special stains (for both coccidia and microsporidia) be performed on concentration sediment to enhance sensitivity (centrifuge for 10 min at 500 Xg).

Currently, there are no FDA approved commercially available immunoassays for detection of the microsporidian spores in clinical specimens. There are a number of different modified trichrome stain methods, some hot and some cold; they all work well, so selection of a particular method often depends on personal preferences among laboratory personnel. Again, it is important to perform these stains on concentrated fecal sediment. Remember that other clinical specimens can also be stained (sputum, urine, etc.), and may contain less debris than that seen in a fecal specimen.

References:

  1. Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.
  2. Isenberg, H. D. (ed.). 2010. Clinical Microbiology Procedures Handbook, 3rd Ed., Vol. 1-3, ASM Press, Washington, D.C.